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1.
Pest Manag Sci ; 77(1): 138-147, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32652887

RESUMO

BACKGROUND: Bursaphelenchus xylophilus is one of the most destructive invasive species, causing extensive economic losses worldwide. The sex ratio of female to male of B. xylophilus plays an important role in the nematode infestation. However, little is known about the processes of its sex determination. The double sex/mab-3-related family of transcription factors are highly conserved in animals, playing crucial roles in sex determination, spermatogenesis and ontogenesis. We therefore investigated its orthologue, Bxy-mab-3, in B. xylophilus. RESULTS: Bxy-mab-3 has two typical conserved DNA-binding domains. It was observed in J2 (the second-stage of juveniles), J3, J4 and male adults (specifically on the spicules), but not in eggs or female adults via mRNA in situ hybridization. RNA-Seq indicated significantly higher expression in males. RNAi showed that the body size and sperm size of male adults were markedly smaller than those of the controls. Meanwhile, almost all the RNAi-treated males failed to mate with the normal females, even 26.34% of interfered males did not produce sperm. However, RNAi of Bxy-mab-3 had no effect on the sex ratio of B. xylophilus. CONCLUSION: Bxy-mab-3 is indispensable for spermatogenesis, ontogenesis and mating behavior. It is a typical sex-determination gene with differential expression in males and females. However, knocking down Bxy-mab-3 expression could not alter the sex ratio as seen in other species. Our findings contribute towards a better understanding of the molecular events of Bxy-mab-3 in B. xylophilus, which provides promising hints for control of pine wilt disease by blocking ontogenesis and decreasing nematode fecundity.


Assuntos
Pinus , Rabditídios , Tylenchida , Animais , Feminino , Masculino , Espermatogênese , Tylenchida/genética
2.
Int J Mol Sci ; 21(3)2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32024175

RESUMO

: Bursaphelenchus xylophilus is a nematode species that has damaged pine trees worldwide, but its pathogenesis has not been fully characterized. α-pinene helps protect host species during the early B. xylophilus infection and colonization stages. In this study, we identified potential molecular mimicry proteins based on a comparative transcriptomic analysis of B. xylophilus. The expression levels of three genes encoding secreted B. xylophilus proteins were influenced by α-pinene. We cloned one gene encoding a thaumatin-like protein, Bx-tlp-2 (accession number MK000287), and another gene encoding a cysteine proteinase inhibitor, Bx-cpi (accession number MK000288). Additionally, α-pinene appeared to induce Bx-tlp-1 expression, but had the opposite effect on Bx-cpi expression. An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees revealed that the α-pinene content was consistent with the expression levels of Bx-tlp-1 (Bx-cpi) and Pm-tlp (Pm-cpi) over time. Thus, these genes likely have important roles contributing to the infection of pine species by B. xylophilus. The results of this study may be relevant for future investigations of the functions of Bx-tlp-1, Bx-tlp-2 and Bx-cpi, which may provide a point to explore the relationship between B. xylophilus and host pines.


Assuntos
Monoterpenos Bicíclicos/farmacologia , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita/genética , Mimetismo Molecular , Pinus/parasitologia , Doenças das Plantas/parasitologia , Tylenchida/metabolismo , Animais , Proteínas de Helminto/genética , Filogenia , Transcriptoma , Tylenchida/efeitos dos fármacos , Tylenchida/genética
3.
PLoS One ; 15(1): e0227476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935232

RESUMO

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a causative agent of pine wilt disease (PWD). To date, although several molecular diagnostic methods have been developed, rapid on-site diagnostic tools for detecting PWN in pinewood are limited. In this study, a point of care diagnostic (POCD) method for detecting PWN in pinewood using recombinase polymerase amplification (RPA) assay was developed. This method comprises quick gDNA extraction buffer (DAP buffer) for the direct extraction of gDNA of PWN from pinewood and a battery-mounted portable optical isothermal device (POID) for the detection of PWD in the field. The RPA assay can distinguish between the PWN and its conspecies which exist in pinewood and can complete diagnostic procedures within 25 min in the field. Moreover, the RPA assay can detect PWN in old wood samples in both natural and stored conditions. The POCD-RPA assay to detect PWN will be useful for epidemiological investigations in the field as well as for quarantine processes in the wood trade.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Pinus/parasitologia , Doenças das Plantas/parasitologia , Tylenchida/genética , Animais , Sequência de Bases , DNA de Helmintos/metabolismo , Genoma Helmíntico , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Recombinases/metabolismo , Alinhamento de Sequência , Tylenchida/isolamento & purificação
4.
PLoS One ; 14(10): e0224391, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31652297

RESUMO

Radopholus similis, commonly known as the burrowing nematode, is an important pest of myriad crops and ornamentals including banana (Musa spp.) and Citrus spp. In order to characterize the potential role of putative effectors encoded by R. similis genes we compared predicted proteins from a draft R. similis genome with other plant-parasitic nematodes in order to define the suite of excreted/secreted proteins that enable it to function as a parasite and to ascertain the phylogenetic position of R. similis in the Tylenchida order. Identification and analysis of candidate genes encoding for key plant cell-wall degrading enzymes including GH5 cellulases, PL3 pectate lyases and GH28 polygalactouranase revealed a pattern of occurrence similar to other PPNs, although with closest phylogenetic associations to the sedentary cyst nematodes. We also observed the absence of a suite of effectors essential for feeding site formation in the cyst nematodes. Clustering of various orthologous genes shared by R. similis with other nematodes showed higher overlap with the cyst nematodes than with the root-knot or other migratory endoparasitic nematodes. The data presented here support the hypothesis that R. similis is evolutionarily closer to the cyst nematodes, however, differences in the effector repertoire delineate ancient divergence of parasitism, probably as a consequence of niche specialization. These similarities and differences further underscore distinct evolutionary relationships during the evolution of parasitism in this group of nematodes.


Assuntos
Genômica , Nematoides/classificação , Filogenia , Tylenchida/classificação , Tylenchida/genética , Animais , DNA Ribossômico/genética , Homologia de Sequência do Ácido Nucleico
5.
Phytopathology ; 109(11): 1949-1956, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31573422

RESUMO

The pine wood nematode Bursaphelenchus xylophilus is a destructive species affecting pine trees worldwide; however, the underlying mechanism leading to pathogenesis remains unclear. In this study, a B. xylophilus gene encoding thaumatin-like protein-1 (Bx-tlp-1) was silenced by RNA interference to clarify the relationship between the Bx-tlp-1 gene and pathogenicity. The in vitro knockdown of Bx-tlp-1 with double-stranded RNA (dsRNA) decreased B. xylophilus reproduction and pathogenicity. Treatments with dsRNA targeting Bx-tlp-1 decreased expression by 90%, with the silencing effect maintained even in the F3 offspring. Pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA decreased the symptom of wilting, and the disease severity index was 56.7 at 30 days after inoculation. Additionally, analyses of the cavitation of intact pine stem samples by X-ray microtomography revealed that the xylem cavitation area of pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA was 0.46 mm2 at 30 days after inoculation. Results from this study indicated that the silencing of Bx-tlp-1 has effects on B. xylophilus fitness. The data presented here provide the foundation for future analyses of Bx-tlp-1 functions related to B. xylophilus pathogenicity.


Assuntos
Pinus , Tylenchida , Virulência , Animais , Técnicas de Silenciamento de Genes , Pinus/parasitologia , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , RNA de Cadeia Dupla , Tylenchida/genética , Tylenchida/patogenicidade , Virulência/genética
6.
Acta Biochim Biophys Sin (Shanghai) ; 51(10): 1071-1078, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31559428

RESUMO

Pine wilt disease, caused by the pine wood nematode Bursaphelenchus xylophilus, leads to severe damage to pine forests in China. In our previous study, effectors secreted by this pathogen were shown to play roles in the different infection stages of pine wilt disease, and a series of candidate effectors were predicted by transcriptome sequencing. This study identified and characterized a novel effector, BxSapB3, which was among these candidate effectors. Agrobacterium-mediated transient expression was used to identify BxSapB3. BxSapB3 was secreted by B. xylophilus and found to be capable of inducing cell death in Nicotiana benthamiana. Quantitative real-time PCR (qRT-PCR) analysis revealed that BxSapB3 was upregulated in a highly virulent strain of B. xylophilus and expressed at lower levels in a weakly virulent strain at the early stages of infection. When BxSapB3 was silenced in B. xylophilus, the process of infection was delayed. These results indicate that BxSapB3 acts as an effector and contributes to virulence at the early stages of B. xylophilus infection.


Assuntos
Proteínas de Helminto/genética , Pinus/parasitologia , Doenças das Plantas/parasitologia , Tylenchida/genética , Animais , Expressão Gênica , Interferência de RNA , Tylenchida/patogenicidade , Fatores de Virulência/genética
7.
Plant Dis ; 103(11): 2851-2856, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31486741

RESUMO

Foliar nematodes represent a minor feeding group within the genus Aphelenchoides Fischer, 1894. The facultative plant parasitic species A. blastophthorus can cause crinkling of leaves, reduced vigor, and stunting of agricultural and ornamental plants. Here we report the first finding of A. blastophthorus in leaves, crowns, and roots of strawberry plants collected in Switzerland in 2018. Species identification was confirmed by morphological and morphometric characterization supported by molecular barcoding of 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), and cytochrome c oxidase I (COI) gene fragment analyses. Phylogenetic analysis of 18S indicated that A. blastophthorus was grouped within close distance to A. fragariae, a well-known foliar nematode affecting strawberry plants. Furthermore, the newly generated molecular barcodes of the partial 28S and COI of A. blastophthorus will support species identification in the future.


Assuntos
Fragaria , Tylenchida , Animais , Código de Barras de DNA Taxonômico , Fragaria/parasitologia , Genes de Helmintos/genética , Filogenia , Suíça , Tylenchida/anatomia & histologia , Tylenchida/classificação , Tylenchida/genética
8.
Int J Mol Sci ; 20(17)2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484427

RESUMO

Studying sex communication is necessary to develop new methods to control the population expansion of gonochoristic species Bursaphelenchus xylophilus, the pathogen of pine wilt disease (PWD). Small chemical signals called ascarosides have been reported to attract potential mates. However, they have not been studied in the sex attraction of B. xylophilus. Here, we confirmed the sex attraction of B. xylophilus using a chemotaxis assay. Then, we cloned the downstream ascaroside biosynthetic gene Bx-daf-22 and explored its function in the sex attraction of B. xylophilus through bioinformatics analysis and RNA interference. The secretions of females and males were the sources of sex attraction in B. xylophilus, and the attractiveness of females to males was stronger than that of males to females. Compared with daf-22 of Caenorhabditis elegans, Bx-daf-22 underwent gene duplication events, resulting in Bx-daf-22.1, Bx-daf-22.2, and Bx-daf-22.3. RNA interference revealed that the attractiveness of female secretions to males increased after all three Bx-daf-22 genes or Bx-daf-22.3 had been interfered. However, the reciprocal experiments had no effect on the attractiveness of male secretions to females. Thus, Bx-daf-22 genes, especially Bx-daf-22.3, may be crucial for the effectiveness of female sex attractants. Our studies provide fundamental information to help identify the specific components and signal pathways of sex attractants in B. xylophilus.


Assuntos
Tylenchida/genética , Animais , Feminino , Masculino , Interferência de RNA , RNA de Cadeia Dupla/genética
9.
Int J Mol Sci ; 20(18)2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31547281

RESUMO

Cytochrome P450 genes are very important for plant-parasitic nematodes to reproduce and to metabolize xenobiotic compounds generated by their host plants. The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes very high annual economic losses by killing large numbers of pine trees across Asia and into Europe. In this study, we used RNA interference (RNAi) to analyze the function of the cyp-33C9 gene of PWN. Our results showed that expression of the cyp-33C9 gene was suppressed successfully after soaking nematodes for 24 h in cyp-33C9 double-stranded RNA (dsRNA). The silencing of the cyp-33C9 gene significantly decreased the feeding, reproduction, oviposition and egg hatch of B. xylophilus. Meanwhile, the migration speed of B. xylophilus in Pinus thunbergii was reduced in the early stages when the cyp-33C9 gene was silenced in the nematodes. Moreover, knockdown of the cyp-33C9 gene in B. xylophilus caused a decrease in pathogenicity to pine trees. These results suggest that the cyp-33C9 gene plays an important role in the reproduction and pathogenicity of B. xylophilus. This discovery identified several functions of the cyp-33C9 gene in B. xylophilus and provided useful information for understanding the molecular mechanism behind pine wilt disease caused by PWN.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Proteínas de Helminto/genética , Pinus/parasitologia , Doenças das Plantas/parasitologia , Interferência de RNA , Tylenchida/genética , Animais , Reprodução , Tylenchida/patogenicidade , Tylenchida/fisiologia
10.
Int J Mol Sci ; 20(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374896

RESUMO

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen responsible for pine wilt disease (PWD), a devastating forest disease with a pathogenic mechanism that remains unclear. Autophagy plays a crucial role in physiological and pathological processes in eukaryotes, but its regulatory mechanism and significance in PWN are unknown. Therefore, we cloned and characterized three autophagy genes, BxATG5, BxATG9, and BxATG16, in PWN. BxATG9 and BxATG16 were efficiently silenced through RNA interference, and we found that BxATG16 positively regulated the expression of BxATG5. Silencing BxATG9 and BxATG16 severely inhibited feeding and reproduction in PWN, indicating that autophagy is essential for these processes. We then examined the expression patterns of these three autophagy genes in PWN under the stresses of α-pinene and H2O2, the main defense substances of pine trees, and during the development of PWD using quantitative reverse transcription polymerase chain reaction. The expression levels of BxATG5, BxATG9, and BxATG16 all significantly increased after nematodes were stressed with α-pinene and H2O2 and inoculated into pine trees, suggesting that autophagy plays an important role in the defense and pathogenesis of PWN. In this study, the molecular characteristics and functions of the autophagy genes BxATG5, BxATG9, and BxATG16 in PWN were elucidated.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Proteínas de Helminto/genética , Pinus/parasitologia , Doenças das Plantas/parasitologia , Tylenchida/genética , Animais , Autofagia , Proteína 5 Relacionada à Autofagia/genética , Regulação da Expressão Gênica , Tylenchida/fisiologia
11.
Sci Rep ; 9(1): 11788, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409860

RESUMO

The root-knot nematodes of the genus Meloidogyne are highly adapted, obligate plant parasites, consisting of nearly one hundred valid species, and are considered the most economically important group of plant-parasitic nematodes. Six Meloidogyne species: M. arenaria, M. hapla, M. incognita, M. microtyla, M. naasi and M. nataliei were previously reported in Michigan, USA. For this study, Meloidogyne nataliei was isolated from the grapevine Vitis labrusca from the type locality in Michigan, USA, and was characterized using isozyme analysis and ribosomal and mitochondrial gene sequences. No malate dehydrogenase activity was detected using macerate of one, five, six, seven or ten females of M. nataliei per well. However, one strong band (EST = S1; Rm: 27.4) of esterase activity was detected when using homogenates of ten egg-laying females per well. Phylogenetic analyses of sequences of the partial 18S ribosomal RNA, D2-D3 of 28S rRNA, internal transcribed spacer of rRNA, mitochondrial cytochrome oxidase subunit I genes and the cytochrome oxidase subunit II-16S rRNA intergeneric fragment from fifty-five valid Meloidogyne species and M. nataliei were conducted using Bayesian inference and maximum likelihood methods. From these results, we infer 11 distinct clades among studied species, with M. nataliei and M. indica composing a basal lineage. Seventy five percent of these species belong to seven clades within the Meloidogyne superclade. Characterization of these clades is provided and evolutionary trends within the root-knot nematodes are discussed.


Assuntos
Evolução Molecular , Família Multigênica/genética , Tylenchida/genética , Tylenchoidea/genética , Animais , Teorema de Bayes , Análise por Conglomerados , Filogenia , RNA Ribossômico 16S/genética , Tylenchida/classificação , Tylenchoidea/classificação
12.
Philos Trans R Soc Lond B Biol Sci ; 374(1767): 20180323, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30967022

RESUMO

Reactive oxygen species (ROS) play important roles in defence response of host plants versus pathogens. While generation and detoxification of ROS is well understood, how varied ability of different isolates of pathogens to overcome host ROS, or ROS contribution to a particular isolate's pathogenicity, remains largely unexplored. Here, we report that transcriptional regulation of the ROS pathway, in combination with the insulin pathway, increases the pathogenicity of invasive species Bursaphelenchus xylophilus. The results showed a positive correlation between fecundity and pathogenicity of different nematode isolates. The virulent isolates from introduced populations in Japan, China and Europe had significantly higher fecundity than native avirulent isolates from the USA. Increased expression of Mn-SOD and reduced expression of catalase/ GPX-5 and H2O2 accumulation during invasion are associated with virulent strains. Additional H2O2 could improve fecundity of Bu. xylophilus. Furthermore, depletion of Mn-SOD decreased fecundity and virulence of Bu. xylophilus, while the insulin pathway is significantly affected. Thus, we propose that destructive pathogenicity of Bu. xylophilus to pines is partly owing to upregulated fecundity modulated by the insulin pathway in association with the ROS pathway and further enhanced by H2O2 oxidative stress. These findings provide a better understanding of pathogenic mechanisms in plant-pathogen interactions and adaptive evolution of invasive species. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.


Assuntos
Expressão Gênica , Estresse Oxidativo/fisiologia , Doenças das Plantas/parasitologia , Tylenchida/fisiologia , Tylenchida/patogenicidade , Animais , China , Europa (Continente) , Árvores/parasitologia , Tylenchida/genética , Estados Unidos , Virulência/fisiologia
13.
Sci Rep ; 9(1): 6080, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988401

RESUMO

The pine wood nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease, one of the most devastating forest diseases in East Asian and West European countries. The lifecycle of B. xylophilus includes four propagative larval stages and gonochoristic adults which are involved in the pathogenicity, and two stages of dispersal larvae involved in the spread of the disease. To elucidate the ecological roles of each developmental stage in the pathogenic life cycle, we performed a comprehensive transcriptome analysis using RNA-seq generated from all developmental stages of B. xylophilus and compared transcriptomes between stages. We found more than 9000 genes are differentially expressed in at least one stage of the life cycle including genes involved in general nematode biology such as reproduction and moulting but also effector genes likely to be involved in parasitism. The dispersal-stage transcriptome revealed its analogy to C. elegans dauer and the distinct roles of the two larval stages from each other regarding survival and transmission. This study provides important insights and resources to understand B. xylophilus parasitic biology.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Estágios do Ciclo de Vida/genética , Pinus/parasitologia , Doenças das Plantas/parasitologia , Tylenchida/genética , Distribuição Animal , Animais , Genes de Helmintos/genética , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , RNA-Seq , Tylenchida/patogenicidade
14.
Int J Mol Sci ; 20(1)2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30626082

RESUMO

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.


Assuntos
Catepsina L/genética , Pinus/parasitologia , Tylenchida/crescimento & desenvolvimento , Tylenchida/patogenicidade , Animais , Catepsina L/metabolismo , Comportamento Alimentar , Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Doenças das Plantas/parasitologia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodução/genética , Análise de Sequência de DNA , Tylenchida/enzimologia , Tylenchida/genética
15.
Acta Biochim Biophys Sin (Shanghai) ; 51(3): 254-262, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668628

RESUMO

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating disease for Pinus spp. The virulence and resilience of PWN are closely linked to the spread and development of PWD. Numerous studies have shown that autophagy has important physiological and pathological functions in eukaryotes. But little is known about the relationships between autophagy and PWNs' virulence and resistance. In this study, through observation under the microscope and recording, we found the induction of autophagy by rapamycin could dramatically improve movement ability of PWNs with different virulence, and the highly virulent AMA3 isolate moved more than the low virulent YW4 isolate when autophagy was over-induced. High concentrations of rapamycin substantially improved the feeding and reproduction of AMA3 but not YW4. Conserved domains of autophagy genes BxATG3, BxATG4, and BxATG7 were first cloned from PWNs by reverse transcription-polymerase chain reaction (RT-PCR). Expression profiling of these three autophagy genes under biotic and abiotic stresses in PWNs with different virulence was determined by quantitative RT-PCR. The results revealed the expression levels of these three autophagy genes in PWNs with different virulence were increased significantly when nematodes were subject to high and low temperatures, oxidative stress, and defensive responses of pine trees. The expression levels of autophagy genes under biotic and abiotic stresses in AMA3 were higher than those in YW4, and different genes showed different performance. Our study clarified that autophagy was closely related to virulence and resistance of PWN, and the ability of a highly virulent isolate to regulate autophagy activity under stresses was stronger than that of a low virulent isolate.


Assuntos
Autofagia/genética , Pinus/parasitologia , Doenças das Plantas/parasitologia , Sirolimo/farmacologia , Estresse Fisiológico , Tylenchida/efeitos dos fármacos , Animais , Estresse Oxidativo , Temperatura , Tylenchida/genética , Tylenchida/patogenicidade , Tylenchida/fisiologia , Virulência
16.
Mol Plant Microbe Interact ; 32(4): 452-463, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30351223

RESUMO

The pine wood nematode (PWN) Bursaphelenchus xylophilus has caused serious damage to pine forests in China. Effectors secreted by phytonematodes play a role in host infection. We identified and characterized an effector, BxSapB1, based on the B. xylophilus transcriptome at the early stages of infection and the transient expression of proteins in Nicotiana benthamiana. BxSapB1 triggered cell death in N. benthamiana when secreted into the apoplast, and this effect was independent of N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1) and suppressor of BIR1-1 (NbSOBIR1). The signal peptide of BxSapB1 was proven to be functional in yeast using the yeast signal sequence trap system and BxSapB1 was strongly expressed in the subventral gland cells of B. xylophilus, as revealed by in-situ hybridization. In addition, based on local BLAST analysis, the BxSapB1 showed 100% identity to BUX.s00139.62, which was identified from the B. xylophilus secretome during Pinus thunbergii infection. BxSapB1 was upregulated in a highly virulent strain and downregulated in a weakly virulent strain of PWN at the early stages of infection. RNA interference assays showed that silencing BxSapB1 resulted in decreased expression of pathogenesis-related genes (PtPR-1b, PtPR-3, and PtPR-5) as well as delayed onset of symptoms in P. thunbergii infected by B. xylophilus. The combined data suggest that BxSapB1 can trigger cell death in N. benthamiana and that it contributes to the virulence in B. xylophilus during parasitic interaction.


Assuntos
Pinus , Tylenchida , Virulência , Animais , Morte Celular , China , Pinus/parasitologia , Tylenchida/genética , Tylenchida/patogenicidade , Virulência/genética
17.
J Helminthol ; 94: e16, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30486905

RESUMO

A new species of Paurodontoides, P. siddiqii n. sp., is described and illustrated based on its morphological, morphometric, and molecular characters. The new species is characterized by a female 550-729 µm long, lip region continuous with body contour, stylet length 7.0-8.0 µm long or c. 1.0-1.2 times the lip region diameter, lateral fields with four smooth incisures, excretory pore at 85-125 µm from anterior end located at the base of the pharyngeal bulb or posterior to it, basal pharyngeal bulb with a short posterior extension projecting into the intestine, monodelphic-prodelphic reproductive system with prominent 19-22 µm long post-uterine sac, and elongate conoid tail with a filiform terminus. The new species is compared with two known species of the genus. It differs from the type species of the genus, P. linfordi, by having slightly shorter stylet, lateral field with smooth incisures, different position of the excretory pore, and absence of male. Compared to P. latus, the new species has a shorter body, shorter stylet, different position of the excretory pore, female tail shape and absence of male. The new species was also compared with close species of the genus Paurodontus because of lateral field marked with four lines, asymmetrical stylet knobs and absence of male. Molecular phylogenetic studies of the new species using partial sequences of 18S rDNA revealed that it forms a clade with a species of the genus Ficotylus. In phylogenetic analyses using partial sequences of the 28S rDNA D2-D3 domain, the new species formed a monophyletic group with a species of the genus Veleshkinema and Sphaerularia spp. (Sphaerulariinae).


Assuntos
Genitália/crescimento & desenvolvimento , Pinus/parasitologia , Casca de Planta/parasitologia , Tylenchida/crescimento & desenvolvimento , Tylenchida/genética , Animais , Tamanho Corporal , DNA de Helmintos/genética , DNA Ribossômico/genética , Feminino , Genitália/anatomia & histologia , Irã (Geográfico) , Masculino , Filogenia , Tylenchida/classificação , Tylenchida/isolamento & purificação
18.
PLoS One ; 13(11): e0202841, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30496181

RESUMO

The study of parasites in recently established populations of invasive species can shed light on the sources of invasion and possible indirect interactions between the alien species and native ones. We studied parasites of the global invader Harmonia axyridis (Coleoptera: Coccinellidae) in the Caucasus. In 2012, the first established population of Ha. axyridis was recorded in the Caucasus in Sochi (south of European Russia, Black Sea coast). By 2018, the ladybird had spread to a vast area: Armenia, Georgia and south Russia (Adygea, the Krasnodar territory, the Stavropol territory, Dagestan, Kabardino-Balkaria and North Ossetia). The examination of 213 adults collected in Sochi in 2018 showed that 53% were infested with Hesperomyces virescens fungi (Ascomycota: Laboulbeniales) and that 8% were infested with Parasitylenchus bifurcatus nematodes (Nematoda: Tylenchida, Allantonematidae). The examined Ha. axyridis specimens were free of the parasitic mite Coccipolipus hippodamiae. An analysis of the phylogenetic relationships of P. bifurcatus based on 18S rDNA confirmed the morphological identification of this species. Hesperomyces virescens and P. bifurcatus were first recorded in the Caucasus and Russia, although they are rather widespread in Europe. This likely indicates that they appeared as a result of coinvasion with their host because the populations of Ha. axyridis, He. virescens and P. bifurcatus in the Caucasus are isolated from the main parts of the ranges of these species in Europe. The nearest localities of Ha. axyridis is on another shore of the Black Sea, and the nearest localities of He. virescens and P. bifurcatus are more than 1000 km from the Caucasus. It is impossible to determine whether the first founders of the Caucasian population were infested with the parasites or whether the parasites were introduced by specimens of Ha. axyridis that arrived later from Europe. Harmonia axyridis was released in the region for pest control, but laboratory cultures are always free of He. virescens and P. bifurcatus. Therefore, the detection of He. virescens and P. bifurcatus indicates that the population of Ha. axyridis in the Caucasus could not have derived exclusively from released specimens. We did not find He. virescens on 400 specimens of 29 other ladybird species collected from the same localities as Ha. axyridis in the Caucasus. No reliable correlation between infestation by He. virescens and that by P. bifurcatus has been found. In addition to these two parasites, an unidentified species of the order Mermithida was recorded. This is the first documented case of Ha. axyridis infestation by a parasitic nematode of this order in nature.


Assuntos
Ascomicetos , Besouros/microbiologia , Besouros/parasitologia , Filogenia , Tylenchida , Animais , Armênia , Ascomicetos/classificação , Ascomicetos/genética , Besouros/genética , Georgia , Federação Russa , Tylenchida/classificação , Tylenchida/genética
19.
BMC Genomics ; 19(1): 553, 2018 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-30053797

RESUMO

BACKGROUND: Plant-parasitic nematodes cause severe damage to a wide range of crop and forest species worldwide. The migratory endoparasitic nematode, Bursaphelenchus xylophilus, (pinewood nematode) is a quarantine pathogen that infects pine trees and has a hugely detrimental economic impact on the forestry industry. Under certain environmental conditions large areas of infected trees can be destroyed, leading to damage on an ecological scale. The interactions of B. xylophilus with plants are mediated by secreted effector proteins produced in the pharyngeal gland cells. Identification of effectors is important to understand mechanisms of parasitism and to develop new control measures for the pathogens. RESULTS: Using an approach pioneered in cyst nematodes, we have analysed the promoter regions of a small panel of previously validated pharyngeal gland cell effectors from B. xylophilus to identify an associated putative regulatory promoter motif: STATAWAARS. The presence of STATAWAARS in the promoter region of an uncharacterized gene is a predictor that the corresponding gene encodes a putatively secreted protein, consistent with effector function. Furthermore, we are able to experimentally validate that a subset of STATAWAARS-containing genes are specifically expressed in the pharyngeal glands. Finally, we independently validate the association of STATAWAARS with tissue-specific expression by directly sequencing the mRNA of pharyngeal gland cells. We combine a series of criteria, including STATAWAARS predictions and abundance in the gland cell transcriptome, to generate a comprehensive effector repertoire for B. xylophilus. The genes highlighted by this approach include many previously described effectors and a series of novel "pioneer" effectors. CONCLUSIONS: We provide a major scientific advance in the area of effector regulation. We identify a novel promoter motif (STATAWAARS) associated with expression in the pharyngeal gland cells. Our data, coupled with those from previous studies, suggest that lineage-specific promoter motifs are a theme of effector regulation in the phylum Nematoda.


Assuntos
Regiões Promotoras Genéticas , Tylenchida/genética , Animais , Motivos de Nucleotídeos , Faringe/metabolismo , Transcriptoma , Tylenchida/metabolismo
20.
J Exp Bot ; 69(12): 3009-3021, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29648636

RESUMO

INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is a signaling peptide that regulates cell separation in Arabidopsis including floral organ abscission and lateral root emergence. IDA is highly conserved in dicotyledonous flowering plant genomes. IDA-like sequences were also found in the genomic sequences of root-knot nematodes, Meloidogyne spp., which are globally deleterious pathogens of agriculturally important plants, but the role of these genes is unknown. Exogenous treatment of the Arabidopsis ida mutant with synthetic peptide identical to the M. incognita IDA-like 1 (MiIDL1) protein sequence minus its N-terminal signal peptide recovered both the abscission and root architecture defects. Constitutive expression of the full-length MiIDL1 open reading frame in the ida mutant substantially recovered the delayed floral organ abscission phenotype whereas transformants expressing a construct missing the MiIDL1 signal peptide retained the delayed abscission phenotype. Importantly, wild-type Arabidopsis plants harboring an MiIDL1-RNAi construct and infected with nematodes had approximately 40% fewer galls per root than control plants. Thus, the MiIDL1 gene produces a functional IDA mimic that appears to play a role in successful gall development on Arabidopsis roots.


Assuntos
Proteínas de Arabidopsis/análise , Arabidopsis/parasitologia , Regulação da Expressão Gênica de Plantas , Proteínas de Helminto/genética , Doenças das Plantas/parasitologia , Tylenchida/fisiologia , Animais , Arabidopsis/genética , Proteínas de Helminto/metabolismo , Tylenchida/genética
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